Chemoresponsive sensors have numerous medical, environmental, and defense applications (Ramsay (ed.) Commercial Biosensors: Applications to Clinical, Bioprocess, and Environmental Samples (John Wiley & Sons, New York (1998)). One of the main challenges in sensor development is devising materials combining analyte binding diversity with mechanisms that transduce molecular recognition events (Ellis et al, Chem. Rev. 100:2477–2478 (2000), Hellinga et al, Trends Biotechnol. 16:183–189 (1998)). Bioelectronic interfaces (Wilner et al, Agnew. Chem. mt. Ed 39:1180–1218 (2000), Ottovaleitmannova et al, Frog. Surf Sci. 41:337–445 (1992), Gopel, Biosensors Bioelect. 10:35–59 (1995)) provide a potentially powerful approach for the development of such devices. These consist of chimeric materials in which a biological macromolecule is assembled on a conducting support, and ligand binding is coupled to an electronic response (Heller, J. Phys. Chem. 96:3579–3587 (1992), Birge et al, J. Phys. Chem. B 103:10746–10766 (1999), Katz et al, Angew Chem. mt. Ed 37:3253–3256 (1998), Wilner et al, J. Am. Chem. Soc. 121:6455–6468 (1999)). Few successful bioelectronic sensors have been developed (Boon et al, Nat. Biotechnol. 18:1096–1100 (2000), Cornell et al, Nature 387:580–583 (1997)), however, because most proteins lack the functionalities to establish ligand-mediated electronic communication.
Proteins that allosterically link the behavior of two different sites do so via conformational coupling mechanisms (Perutz, Mechanisms of Cooperativity and Allosteric Regulation in Proteins (Cambridge University Press, Cambridge) 1990). In such proteins, two sites are thermodynamically coupled when each adopts multiple, distinct local conformations that correspond to distinct global protein conformations. Such global conformational changes often correspond to different quarternary states in multimeric assemblies (Gerstein et al, Biochemistry 33:6739 (1994)) but may also involve motions such as ligand-induced hinge-bending motions (Gerstein et al, Biochemistry 33:6739 (1994)) within monomers. Such motions are found in many proteins (Gerstein et al, Biochemistry 33:6739 (1994)) and are common to all structurally characterized members of the bacterial periplasmic binding protein (bPBP) superfamily (Tam et al, Microbiol. Rev. 57:320–346 (1993)). These proteins have similar overall structures consisting of a single chain that folds into two domains linked by a hinge region (Fukami-Kobayashi et al, J. Md. Biol. 286:279–290 (1999), Quiocho et al, Mol. Microbiol. 20:17–25 (1996)).
The present invention results, at least in part, from studies demonstrating that it is possible to couple ligand binding in bPBPs to modulation of the interactions between a redox reporter group and a modified electrode surface. This scheme is analogous to ligand-dependent allosteric control of intermolecular macromolecular associations as observed in electron transport chains (Georgiadis et al, Science 257:1653 (1992); Iwata et al, Science 281:64 (1998)) and provides the basis for powerful bioelectronic sensors.